GB 135_miniPERM.indd
Contents
1. SS UM 350 400 ml medium SARSTEDT 42 SARSTEDT 43 Technical modifications reserved GB 135 0413 0413 User manual miniPERM SARSTEDT AG amp Co P O Box 12 20 D 51582 N mbrecht Phone 49 2293 3050 Fax 49 2293 305 122 info sarstedt com www sarstedt com SARSTEDT2. User Manual miniPERM When the cell suspension Is introduced the air displaced by the medium must be released through one of the other ports Therefore a second screw cap should be loosened Medium at the rim A of the filling port must be thoroughly removed by drawing up with an exhauster before closing the port 3 Filling the miniPERM bioreactor 3 1 Introducing the cell suspension into the production module The cell suspension is introduced into the production module with a syringe through one of the three ports equipped with Luer Lock connectors e Mount the miniPERM bioreactor on the stand with the production module on top so that one of the three Luer Lock connectors is positioned at the highest point fig 5 Draw up the prepared cell suspension 5 35 or 50 ml through a sterile filling tube 5 using a disposable syringe 50 ml Remove air bubbles from the syringe before injecting the cell suspension fig 6 Remove the screw cap at the uppermost point A Screw the syringe without the filling tube onto the Luer Lock connector fig 7 Turn the miniPERM bioreactor so that one of the other screw caps is positioned at the highest point Loosen a second screw cap at the uppermost point C Inject the cell suspension slowly into the production module fig 8 Close the Luer Lock connector of the port at position C tightly with a sc
3. Duration of the miniPERM culture Leakage 1 Between production and nutrient module During assembly of the miniPERM the snapper clips on the production module are snapped audlibly into the indentations on the rim of the nutrient module Do not attempt to twist the two modules together 2 At the screw cap of the neck opening of the nutrient module Pressure The silicone membrane of the production module is always expanded outwards due to a build up of pressure in the bioreactor The silicone membrane of the production module is pulled inward SARSTEDT 36 e his depends on the growth kinetics of the cell clone By regularly changing the medium and regularly harvesting the cells the miniPERM bioreactor culture can be operated in a continuous way over several weeks The timing of the medium exchange and cell harvesting will vary from cell line to cell line e After assembly ensure that all the snapper clips are fully snapped with their corresponding indentations on the rim of the nutrient module A second check will ensure that nutrient and production modules are located correctly e he reusable nutrient module should be autoclaved to a maximum temperature of 121 C It should be used no more than 10 times otherwise this leads to deformation and cracking of the polycarbonate e o avoid damage to the polycarbonate the nutrient module should be placed into the autoclave In a way as to be contact free f
4. User Manual miniPERM Se SARSTEDT User Manual miniPERM User Manual miniPERM Contents AW OGIIGHION ve a ee er I ee SA 26 Design and function of the miniPERM bioreactor OBEN Mle Sige Gil eg connect ee ere eee eee 28 Example of hybridoma cell culture High density cell culture in the miniPERM biOre CctlOf en ee 34 High density cell culture Oxygen requirement of hybridoma cells Production rate Changing the medium Handling of the miniPERM bioreactor CUBE NOG aaa ee AE PEER ee 35 Problems and solutions IE EINE ee EE ee 39 Publications and application notes OCZ GZ 40 Ordering Mionna EM 41 BET DIS FUSION aE A E NT 42 SARSTEDT SARSTEDT 24 25 User Manual miniPERM 1 Introduction Design of the miniPERM bioreactor miniPERM is a bioreactor made of two connected modules designed for the cell culture of hybridoma and other cells in high densities high density culture It consists Of e a disposable production module which serves as a culture chamber with a volume of 35 ml or 50 ml miniPERM classic or HDC 50 and e an autoclavable reusable or sterile disposable nutrient module made of plastic polycarbonate which serves as a media reservoir with 550 ml volume that is attached to the production module To separate the two modules the production module is fitted with a semi permeable dialysis membrane MWCO 12 5 kDa on the side facing the nutrient mod ule when th
5. An air Space must be maintained above the nutrient medium in the bioreactor e In the miniPERM classic some adherent cells e g CHO grow in the suspension in an aggregate form This has no influence on the productivity of the cells e Use of the miniPERM SM production module This module has 2 integrated stationary matrices made of polyester e Use of Microcarrier beads for the production of secreted recombinant proteins The cells grow on the microcarrier in the miniPERM classic production module e Cells of some lines do not grow to high densities less than 2 0 x 10 cells ml If it is possible to cultivate the cells at average cell densities and high viability over a long time period by regular changes of the medium in the nutrient module high antibody concentration can be obtained e The cells must always be kept in suspension If the miniPERM bioreactor is removed from the Universal Turning Device and placed in the sterile hood the cells will immediately begin to settle In this case they may form clusters and possibly die To avoid such an occurrence all necessary manipulations must be carried out as quickly as possible e Use an antifoam agent e g antiFOAM Ordering Information see page 41 Add 0 5 to 1 ml to the nutrient module e Reduce the serum concentration in the medium contained in the nutrient module and if possible also in the production module SARSTEDT 35 User Manual miniPERM
6. The oxygen consumption rate of hybridoma cells is in the order of 5ug per 10 cells per hour With a cell density of 107 cells ml which is easily attainable in the miniPERM bioreactor the oxygen requirement of the 35 x 107 cells cultivated in the 35 ml miniPERM classic of the production module is approximately 1 75 mg hour Production rate To produce a certain amount of product monoclonal antibodies in the case of hybridoma cells a certain number of cells are needed The number of cells required depends on the individual properties of the cells cultured According to Fazekas de St Groth J Immunol Methods 57 1983 121 hybridoma cells typically produce between 4 x 10 and 7 x 10 antibody molecules per cell in a 24 hour period Changing medium The stressful conditions of high density cell culture means the cells require a medium of particularly high quality with respect to the content of glucose and NaHCO The content of glucose should not be less than 4 g l If less the medium should be supplemented with additional glucose Most media have an NaHCO content DMEM 3 78 g l which ensures a buffer capacity up to 2 weeks Due to the optimal gas exchange in the miniPERM bioreactor and the high cell density the initial glucose content would be exhausted before the pH colour change of the nutrient medium is indicated from red to yellow Thus the buffer capacity of the medium to neutralize and to buffer metabolic wast
7. class Il antigens on human alveolar macrophages by granulocyte macrophage colony stimulating factor in the presence of glucocorticoids Immunology 98 1999 104 110 Falkenberg F W Production of monoclonal antibodies in the miniPERM bioreactor comparison with other hybridoma culture methods Res Immun 6 149 1998 560 570 Falkenberg F W Weichert H Krane M Bartels Palme M Nagels H O Fiebig H In vitro production of monoclonal antibodies in high concentration in a new and easy to handle modular minifermenter J Immun Meth 179 1995 13 29 Kagan E Vieira E Petrie H T Comparison of hollow fiber bioreactors and modular minifermentors for the production of antobodies CAAT OPRR Workshop on Alternatives in Monoclonal Antibody Production 9 1997 Haborplace Hotel Baltimore MD Little M Kipriyanov S M Le Gall F Moldenhauer G Of mice and men hybridoma and recombinant antibodies Review Immunology Today 08 00 364 370 Metzger J Nicklisch N Schmidt B Kufer P Peschel C Bernhard H Induction of a T helper cell response against the tumor associated antigen HER 2 using monocyte derived dendritic cells ESACT Meeting 09 2001 Sweden Raulf Heimsoth M Sander Zhiping Ch Borowitzki G Diewald K van Kampen V Baur X Development of a monoclonal antibody based sandwich ELISA for detection of the latex allergen Hev b Int Arch Allergy Immunol 123 2000 236 2
8. For the production of biomass the medium should be changed in intervals of up to 5 days The medium in the nutrient module should be replaced as soon as there is a slight change in colour from salmon pink to a yellowish pink This signifies that the buffering capacity of the medium is reaching the point of exhaustion Under high density cell culture conditions serum concentration can be critical In the miniPERM bioreactor the serum concentration should be not less than that used in a stationary culture of the same cell line For the production of monoclonal antibodies or other proteins a serum concentration between 5 to 30 should be used For the production of biomass a serum concentration of 10 is generally sufficient In the miniPERM nutrient module the serum concentration can be halved and in some cases it can be reduced to zero It is also possible to cultivate hybridoma or other cell types in the miniPERM bioreactor The cells must be adapted to a serum or protein free environment User Manual miniPERM Publications amp Application Notes Bruce M P Boyd V Duch White J R Dialysis based bioreactor systems for the production of monoclonal antibodies alternatives to ascites production in mice Journal of Immunological Methods 264 No 1 2 06 2002 59 68 Caulfield J J Fernandez M H Sousa A R Lane S J Lee T H Hawrylowicz C M Regulation of major histocompatibility complex
9. a Universal Turning Device during cultivation Due to the exceptionally high cell densities produced in the miniPERM a higher than usual turning speed is required For this reason we recommended the use of the Universal Turning Device which can rotate the miniPERM from 0 1 to 40 rom compared to standard turners that usually have a maximum speed of 2 0 rpm SARSTEDT 27 User Manual miniPERM Operating Instruction All the operations A described below must be carried out in a sterile hoodl Cells are cultivated under normal laboratory conditions centrifuged and adjusted to the specified density Ensure the clips A of the production module are completely snapped It is very important A that the nutrient module is autoclaved to a maximum of 121 C and no more than 10 times Higher temperatures lead to deformation of the polycarbonate Before connecting the two modules unscrew the screw cap of the nutrient module to prevent pressure build up Ensure the clips A of the production module are completely snapped SARSTEDT 28 1 Preparation of cells for culturing in the miniPERM bioreactor using hybridoma cells as example The miniPERM classic culture can be started with about 35 to 37 ml of a cell suspension containing approximately 1 to 5 x 10 cells ml 35 to 185 x 10 cells total fig 1 In most cases the contents of 1 to 2 well grown 150 cm cell culture flas
10. has to be rotated at low speed at the beginning of the culture With an increase of cell density the rolling speed can be increased concomitantly Examples e murine hybridoma cells 5 to 20 rpm Display 10 40 e human hybridoma and transfected cells 2 to 10 rom Display 4 20 The rolling soeeds refer to the display on the Control unit miniPERM actually rotates at half this speed The rollers of the Universal Turning Ensure that the Universal Turning Device is correctly assembled Device stop working e After cleaning the turning device with pharmaceutical grade ethanol or isopropanol you must oil the bearings Do not use n propanol Caution Keep the rubber material of the rollers free from oil in order to prevent the bioreactors from slipping from the device e Do not autoclave the Universal Turning Device SARSTEDT 37 User Manual miniPERM Promem soson Medium Changing the medium Serum SARSTEDT 38 Under conditions of stress in each high density cell culture the cells require a medium of particular high quality with regard to the content of glucose and NaHCO In the miniPERM bioreactor the cells can be cultivated with the same NaHCO dependent media normally used in tissue culture The content of glucose should be not less than 4 g l otherwise the medium should be additionally supplemented accordingly For the production of proteins the medium should be changed in intervals of up to 7 days
11. 1 Single use 50 ml Luer Lock Syringe sterile 8 e Single use 2 ml Luer Syringe sterile 20 e Filling Tube sterile 8 e Luer Syringe Needles 25G x 3 sterile 20 Septum Ports sterile 6 Stand for miniPERM 1 cellPROTECT 1 ml sterile 1 antiFOAM 5 ml sterile 1 Universal Turning Device Packaging 94 6001 061 Universal Turning Device 115 230 V 1 SARSTEDT 41 User Manual miniPERM User Manual miniPERM Brief instructions for use Medium change For example Preparation of cells for cultivation in miniPERM classic production module with 35 ml volume For using the miniPERM HDC 50 production module 1 5 x 10 cells ml in 50 ml medium are prepared Preparation of cells gt gt 350 400 ml medium gt 10 min b 4 Sampling 20 20 20 gt ml ml ml ol 1 5 x 10 cells ml 120 miniPERM preparation The sterile bioreactor option is ready for use without further preparation Autoclavable nutrient module 1 2 bar 121 C 250 F 20 min cil lt gt Filling 600 x g 10 min 4 C
12. 41 8 2000 Sch tt C F rll B Stelter F Jack R S Witt S CHO transfectants produce large amounts of recombinant protein in suspension culture Journal of Immunological Methods 204 1997 99 102 Vollmers H P Dammrich J Wozniak E Bier D M ller Hermelink H K Apoptotic in vitro and in vivo activity of the human monoclonal antibody SC 1 on stomach cancer cells 2nd International Gastric Cancer Congress Munich Germany 04 1997 Konstantinov M Mindova M Gospodinov P Genova P Three Dimensional Bioreactor Cultures A Useful Dynamic Model for the Study of Cellular Interactions N Y Acad Sci 1030 103 115 2004 Dewar V Voet P Denamur F Smal J Industrial Implementation of in Vitro Production of Monoclonal Antibodies ILAR Journal Volume 46 Number 3 2005 Berlin V Rousselle P Production of arecombinantly expressed laminin fragment by HEK293 EBNA cells cultured in suspension in a dialysis Based Bioreactor Protein Expr Purif 48 1 43 48 2006 Application notes Vollmers H P Wozniak E Cultivation of human hybridoma cell line in the miniPERM bioreactor University of W rzburg Germany Tutas M Cultivation of mouse hybridoma cells in the miniPERM bioreactor Cell Diagnostica GmbH Germany Lingnau A Cultivation of IgM producing hybridoma cells in the miniPERM bioreactor VM PRO GmbH Luckenwalde Germany Schliephacke T K ppel S Long term proliferatio
13. Draw the harvest into syringe e Close the second port B with a sterile screw cap e Turn the bioreactor so that the harvesting port A with the attached syringe is positioned at the uppermost point and remove the syringe e he volume extracted should be replaced by fresh medium see 3 1 Depending on the cell density reached the harvested cells could be resuspended in fresh media and reinjected into the production module User Manual miniPERM Cleanliness is of A utmost importance when changing the medium Risk of contamination Care must be taken to ensure that no traces of the medium have been left on the thread of the filling port or run down the sides of the nutrient module Should this occur remove via an exhauster or with a sterile pad soaked with 70 pharmaceutical ethanol Do not flame For disinfectants of A the screw cap do not use ethanol propanol or disinfectants solvents that contain alcohol as their use will impair function of the PTFE membrane Rinse with distilled water only The reusable screw cap can be autoclaved for 20 min to a maximum of 121 C at least 5 times without change to the material For disinfection A use only 70 pharmaceutical grade ethanol or isopropanol for the miniPERM bioreactor the Universal Turning Device the incubator and water bath All other chemicals especially quaternary ammonium complexes m
14. ECT celIPROTECT increases the viscosity of the culture medium and thereby protects the cells against shear For simple and safe cell suspension inoculation sampling and harvesting Sterile filling tube 5 stress cellPROTECT is added to the cell suspension in the production module at concentrations of 0 05 to The sterile filling tube offers a simple solution to cell 0 1 vA suspension transfer e g from centrifuge tubes to single use syringe A filling tube is slightly flexible connects antiFOAM directly to a 50 ml Luer syringe and has a sufficiently large inner diameter to minimize shear stress induced cell damage When serum containing medium is used significant quantities of foam may develop in both modules of the miniPERM By adding 0 5 to 1 ml antiFOAM to the Luer syringe needles 25G x 5 3 For sampling and supplementing the medium in the Start up Support Kit roduction module via septum ports j j P Accessories to inoculate sample and harvest Septum ports Consists of To simplify sampling and supplementing procedures and e Sterile single use 50 ml Luer Lock syringe 8x to further reduce contamination risks a replacement of one or two of the three standard screw caps on the production module with sterile septum ports is possible e Sterile single use 2 ml Luer syringe 20x e Sterile filling tube 5 Luer 8x e Luer syringe needles 25G x s sterile 20x The rubber septum ports
15. ay cause microcracks Do not use n propanol 6 Changing the medium in the nutrient module Replacing the spent medium with fresh medium follows the same procedure as described in 3 2 for the first filling of the nutrient module e Place the miniPERM in a sterile hood e Unscrew the screw cap of the neck opening on the nutrient module e Pour the used medium into a disinfected waste bottle fig 23 e Fill 350 to 400 ml of fresh tempered 37 C medium into the nutrient module fig 24 e Remove pressure in the miniPERM following the same procedure as described in 3 2 for pressure equilipration e Close the neck opening with a sterile screw cap Tighten the screw cap applying a slight pressure only Cleaning and sterilisation of the nutrient module multiple use nutrient module The reusable nutrient module can be autoclaved at least ten times without changes to the material To avoid damage to the polycarbonate the nutrient module should be placed freely i e not subjected to pressure in the autoclave The maximum autoclave temperature is 121 C e he modules are separated at the end of the culture using the hook end of the opener provided to gently prise open the snapper clips The production module can then be easily removed from the nutrient module fig 25 Clean the nutrient module only with hot water and neutral or weak acid or alkaline detergent without rinsing agents Rinse with ample water t
16. e products is exhausted SARSTEDT 34 The interval of the medium change depends on the cell line the medium and the increase of the cell density We suggest 1 to 2 times per week An orange yellow colour of the medium in the production module is indicative of a high cell density gt 107 cells ml Handling of the miniPERM bioreactor The temperature of the nutrient medium should always be brought to 37 C before the module is filled After MINIPERM is filled with nutrient medium and introduced to the incubator the air above the medium heats and expands This expansion can be substantial per 1 C in temperature the increase in volume is 1 273 of the volume at O C Thus the volume increases by about 24 ml if the temperature in the space above the medium in the nutrient module rises from 4 C to 37 C This increase in volume would cause a substantial rise in pressure more than 0 1 bar in the nutrient module of the miniPERM This would lead to expansion of the silicone membrane of the production module There is another effect typical for cultures with NaHCO buffered media in a closed bioreactor which causes a further rise of pressure in the nutrient module When a culture is started there is usually atmospheric air rather than the CO containing incubator atmosphere in the air space above the medium of the nutrient module Therefore the NaHCO in the medium decomposes and the CO Is released into the space above the mediu
17. e two modules are connected The outer part of the production module is made of a thin gas permeable silicone membrane The screw cap of the nutrient module has an integrat ed gas exchange membrane nutrient module silicone sealing ring support grid dialysis membrane N i gt gt silicone ahi Ra membrane roduction P 07 frame module srew Caps srew cap with integrated Universal Turning Device gas exchange membrane Q nutrient module 1157230 V SARSTEDT 26 Function of the miniPERM bioreactor 1 Dialysis membrane Neither the cells nor high molecular weight products released by the cells e g secreted monoclonal anti bodies can pass through the semi permeable dialysis membrane However nutrients glucose amino acids vitamins ions and gases O CO dissolved in the medium can diffuse from the nutrient module into the production module Due to more than a 10 fold excess of nutrient medium the cells are provided with substances necessary for cultivation over a long period of time Simultaneously acidic e g lactic acid toxic e g am monium ions and other low molecular mass products of the cell metabolism diffuse from the production module through the dialysis membrane into the nutrient module where they are diluted and neutralised in the large excess of medium The discharge of CO from the miniPERM is further promoted by the fact
18. en pierced 5 or 6 times Harvesting must be A quick to prevent the cells from settling and clumping Medium at the rim A of the harvest port must be thoroughly removed by drawing up with an exhauster SARSTEDT 32 4 2 Taking samples through a septum One of the three ports can be replaced with a septum cap which maintains the sterile barrier for sample removal e Prepare the miniPERM bioreactor as described in 4 1 e Clean the hole of the septum cap with a pad soaked in 70 pharmaceutical ethanol e Pierce a sterile needle with syringe s 5 ml in the hole of the septum cap e Turn the miniPERM bioreactor so that the septum cap with the attached syringe is positioned below the level of the liquid in the production module fig 21 e Draw the sample into the syringe e Remove the needle from the septum cap e Clean the hole of the septum cap with a sterile pad soaked in 70 pharmaceutical ethanol 5 Harvesting The interval and volume of harvesting depend on the cell density and the amount of product which the cells produce Once or twice a week on an average 3 of supernatant is harvested Harvesting is done essentially the same way as sampling e Prepare the miniPERM bioreactor as described in 4 1 e In order to prevent a negative pressure in the production module whilst drawing up the harvest with a syringe it is necessary to loosen a second screw cap at position B fig 22 e
19. ks are sufficient for this purpose For using the miniPERM HDC 50 production module the cell suspension contains 1 to 5 x 10 cells ml in 50 ml 2 Preparation of the miniPERM bioreactor 2 1 Pre assembled miniPERM sterile In this version the miniPERM has been assembled from the two modules and sterilized This reoresents a single use product It is not autoclavable It can be used without further treatment fig 2 2 2 Multiple use miniPERM The production modules are supplied as disposables in an alu pouch The reusable nutrient module must be sterilized by autoclaving to a maximum of 121 C prior to use e Pack the nutrient module in an autoclaving bag fig 3 and autoclave it without the screw cap for 20 min to a maximum of 121 C also refer to item 7 e he alu pouch of the production module is opened to ensure the production module is resting on the screw caps of the Luer Lock connectors The production module should be left in the plastic pack e he autoclaved nutrient module is now pushed against the sealing ring of the production module figs 4 and 4a until the snapper clips of the production module are snapped audibly into the indentations on the rim of the nutrient module e he plastic pack of the production module can now be removed e The assembled miniPERM is now ready for use 1 2 bar 121 C 250 F 20 min
20. licone membrane of the production module fig 13 e Close the neck opening with the screw cap Tighten the screw cap applying a slight pressure only e Place the miniPERM bioreactor onto the Universal Turning Device and set the speed at the Control Unit on 0 1 to 40 rpm The above rolling speeds refer to the display on the Control Unit miniPERM actually rotates at half this speed User Manual miniPERM Under no A circumstances should the screw caps of the three Luer Lock connectors of the production module be opened when the silicone membrane is extended The excess pressure in the nutrient module would cause the cell suspension to spurt out and be lost In addition the outside of the miniPERM bioreactor would be contaminated Should foam A accumulate in the production module which is often the case when FCS is used as a medium supplement it is difficult to loosen the screw cap of the production module without getting foam into the thread To solve this problem an antiFOAM agent can be used in the nutrient module see Troubleshooting Medium at the rim of the harvest port must be thoroughly removed by drawing up with an exhauster before closing the port 4 Taking samples 4 1 Taking samples through a port of the production module e Place the miniPERM bioreactor in a sterile hood e Ensure that pressure is released Hold the
21. m of the nutrient module The result is a further rise in pressure in the nutrient module and alkalinisation of the medium These differences in overall and CO partial pressure will be levelled off by diffusion of gases through the gas exchange membrane of the screw cap for the nutrient module Consequently the pH value will be readjusted to ohysiological levels within a few hours User Manual miniPERM Troubleshooting KU HET TEN The cells are not growing after a few days the percentage of dead cells is above 70 Oulturing of adherent cells The cells do not reach high densities lt 1 x 107 cells ml Cells aggregate and die Accumulation of foam If too much foaming occurs in the nutrient and production modules it is not possible to release the pressure without getting foam on the screw caps e Increase the initial cell density e Produce the starter cell suspension for the miniPERM culture with ml conditioned medium from a cell culture flask and ml fresh medium e Increase the percentage of the serum supplement in the medium e Decrease the rolling soeed when using sensitive cells e g of human hybridoma cell lines Set the rolling speed to just 0 5 rom e Use of cellPROTECT Ordering Information see page 41 cellPROTECT protects the cells against shear forces Add 0 05 to 0 1 to the medium in the production module e Do not fill more than 400 ml medium into the nutrient module
22. miniPERM bioreactor with one hand applying light pressure to the silicone membrane With the other hand loosen do not unscrew the screw cap of the nutrient module so excess pressure Is released see figs 15 15a Close tightly again Position the miniPERM bioreactor on the stand so that one of the ports of the production module is at the highest point A and remove the screw cap of this connector fig 16 Fit a syringe of suitable size approx 1 ml depending on the sample volume to be taken onto the Luer Lock connector in position A fig 17 Turn the miniPERM bioreactor so that the sampling port A with the attached syringe is positioned below the level of the liquid in the production module fig 17 Draw up the sample into the syringe Before removing the syringe miniPERM bioreactor must be turned so that the sampling port with the attached syringe is positioned at the uppermost point fig 18 After removing the syringe air bubbles should be removed by pressing the silicone membrane with the fingers e Close the Luer Lock connector of the sampling port with a sterile screw cap fig 19 and return the miniPERM bioreactor to the incubator SARSTEDT 31 User Manual miniPERM Small diameter A needles 25G x 8 should be used in order to retain the sterile barrier The septum caps should be replaced after they have be
23. mponents cell suspension medium culture vessels incubator etc for sterility Cleanliness is of utmost importance when taking samples harvesting cells or changing the medium e Use a septum port to take samples Replace one screw cap on the production module with a septum Ordering Information see page 41 e Following sampling or cell harvesting the access ports must be free of culture medium before closing with the screw caps e Always use a new screw cap for each sampling harvest port which has been opened e Use of an antifoam agent e g antifoam Ordering Information see page 41 If foam has accumulated in the production module which is often the case when FCS is used as a medium supplement it is difficult to loosen the screw caps of the production or nutrient mod ules without getting foam into the threads This could be a potential risk of contamination e After exchanging the culture medium the neck opening must be free from any such medium prior to closing with a screw cap e Always use a new screw cap for the nutrient module after changing the medium e f contamination is limited to the nutrient module the culture can be saved Remove the cells from the production module and transfer them to another sterile miniPERM bioreactor 1 In the production module 2 In the nutrient module Rotation speed The turning speed must be selected according to the robustness of the cells e Generally the bioreactor
24. n of CHO cells in the miniPERM bioreactor IOnGen AG In vitro Systems Services GmbH Germany M ller S HEK U293 cells cultivated in suspension in the miniPERM bioreactor Knoll AG Ludwigshafen Germany Prestle J Ott Gebauer S Production of recombinant adenoviruses in the miniPERM bioreactor University of Gottingen Germany Sponaas A Anding P Cultivation of a murine macrophage like cell line K774 in the miniPERM bioreactor Max Planck Institut for Infections Biologie Berlin Germany Dobner T Cultivation of insect cells SF9 cells baculovirus system in the miniPERM bioreactor University of Regensburg Germany Schillo M Meyer N Bentrop J Cultivation of insect cells Drosophila Schneider cells S2 in the miniPERM bioreactor University of Karlsruhe Germany Wuppermann F Cultivation of amurine hybridoma cell line in miniPERM classic and miniPERM HDC 50 bioreactors LCTech GmbH Dorfen Germany SARSTEDT 39 User Manual miniPERM User Manual miniPERM miniPERM Accessories eh 10 gt EEEE 20 4 MI aa The following accessories are available for convenient miniPERM stand RET DUN CENE ON For mounting the miniPERM bioreactor during Sterile single use 50 ml Luer Lock and 2 m N Culation sampling and harvest EUER AyTINgeS cellPROT
25. o completely remove the detergent Do not use an automatic dish washer Autoclave the nutrient module without the screw cap for 20 min to a maximum of 121 C Do not use any alkaline based corrosion protector in autoclave boiler water 8 Cleaning the Universal Turning Device The Universal Turning Device can be disinfected with a cloth soaked in 70 pharmaceutical grade ethanol or isopropanol Do not use n propanol Do not autoclave the Universal Turning Device SARSTEDT User Manual miniPERM High density cell culture in miniPERM High density cell culture The design of the miniPERM bioreactor makes it possible to culture cells to considerably higher densities high density cell culture than in conventional stationary culture in which the oxygenation of the cells can only be achieved via gas exchange through the cap Consequently the cellular products are secreted in much higher concentrations compared to conventional cultures Cells cultured in high density are very much dependent on optimal culture so they are More sensitive to disturbances than cells in conventional stationary culture at densities of 10 to 10 cells ml Due to the high density 107 cells ml and greater the cells are dependent on the continuous supply of large quantities of nutrients and oxygen and on the removal of metabolic waste products and CO Oxygen requirement of hybridoma cells
26. offer the possibility for addition e Septum ports sterile 6x of hid to or removal o samples rom the production miniPERM stand 1X ule usi ile single use syringe needles of sma diameter There is no need to open the module The SEEN 1m FIE 1x septum ports should be replaced after they have been antiFOAM 5 ml sterile 1x pierced 5 or 6 times SARSTEDT 40 nutrient module medium this foam build up is reduced Ordering Information miniPERM classic miniPERM HDC miniPERM SM Vol 35 ml 50 Vol 50 ml 240 cm Bioreactor sterile 94 6001 059 12 94 6077 121 12 94 6077 618 42 Test Kit sterile 94 6077 009 1 94 6077 122 1 94 6077 609 1 Production Module sterile 94 6001 055 12 94 6077 017 1Z 94 6077 616 12 Nutrient Module 94 6001 153 4 4 Bioreactors Start up Support Kit miniPERM Accessories Packaging bag box 94 6001 153 Nutrient Module for miniPERM autoclavable 4 94 6001 054 Stand for miniPERM 4 94 6001 036 Screw Cap for Production Module sterile 6 94 6077 037 Screw Cap for Nutrient Module sterile 16 EY Septum Ports sterile 100 94 6077 135 Luer Syringe Needles 25G x 5 8 0 5 x 16 mm sterile 100 94 6077 136 Single use 2 ml Luer Syringe sterile 100 94 6077 137 Single use 50 ml Luer Lock Syringe sterile 60 94 6077 138 Filling Tube 5 Luer sterile 100 94 6077 320 antiFOAM sterile 100 ml 1 94 6077 041 cellPROTECT sterile 100 ml 1 94 6001 094 Start up support kit Pieces
27. rew cap fig 9 Turn the miniPERM bioreactor back so that the port A to which the syringe has been attached is positioned at the uppermost point again fig 10 Remove the syringe Air bubbles should be removed by pressing the silicone membrane Close the production module by tightly screwing a screw cap or a septum cap see Troubleshooting onto the Luer Lock connector of the third port in position A fig 11 SARSTEDT 29 User Manual miniPERM Do not fill more A than 400 ml into the nutrient module An air space must be maintained above the nutrient medium for successful cultivation The rolling speed must be selected according to the robustness of the cells e g e murine hybridoma cells 5 to 20 rpm e human hybridoma and transfected cells 2 to 10 rpm Since the diameter of the miniPERM bioreactor is twice the size of the rolls of the Universal Turning Device miniPERM rotates at half the set speed SARSTEDT 30 3 2 Introducing the medium into the nutrient module The nutrient module is filled with medium through the large neck opening which is fitted with a screw cap as follows e Remove the screw cap from the neck opening of the nutrient module and pour in 350 to 400 ml of tempered nutrient medium fig 12 e To remove pressure in the miniPERM bioreactor press your hand against the si
28. rom any other item e To sterilize the miniPERM bioreactor use only 70 pharmaceutical grade ethanol or isopropanol DO NOT USE any other chemicals especially quaternary ammonium complexes may cause microcracks e Tighten the screw cap of the neck opening of the nutrient module applying slight pressure only e Autoclave the reusable screw cap separately to a maximum of 121 C and no more than 5 times without change of the material The sterile single use screw caps are not autoclavable e Do not flame the neck opening of the nutrient module and the screw caps This will lead to deformation of the polycarbonate e Each time the nutrient module is opened reclose with a new screw Cap e The humidity inside the CO incubator may be too low Make sure that the humidity is at least 90 e The temperature of the nutrient medium should always be brought to 37 C before the module is filled If the nutrient module has been filled with cold medium 4 C and the miniPERM bioreactor has been placed into the incubator the air above the medium heats up and expands This will lead to expansion of the silicone membrane of the production module e Equilibration of pressure For this loosen the nutrient module screw cap by turn and afterwards tighten applying slight pressure only e his may be due to a bacterial or fungal contamination in the nutrient or production modules User Manual miniPERM Contamination First check the co
29. that CO dissolved in the culture medium both physically and in the form of NaHCO can pass through the dialysis membrane from the production module to the nutrient module Cell product Silicone membrane Dialysis membrane The dialysis membrane is covered with a support grid facing the nutrient module This grid has two functions It protects the dialysis membrane from mechanical damage and provides a means of swirling the nutri ent medium at the dialysis membrane surface leading to an improved diffusion of nutrients and metabolic products User Manual miniPERM 2 Silicone membrane Oxygen requirements are fulfilled by O diffusing from the incubator atmosphere into the production module through the silicone membrane The CO produced by the cells in corresponding quantities leaves the production module via the same route The high CO permeability of the silicone membrane in the production module means the NaHCO in the medium is at equilibrium with the CO in the incubator atmosphere 3 Screw cap with gas exchange membrane The gas exchange membrane in the screw cap of the nutrient module enables equilibration of pressure between the bioreactor and the outer environment 4 Universal Turning Device For optimal supply of nutrients and removal of metabolic waste products through the membranes cells and the medium must be circulated continuously Therefore the miniPERM bioreactor is rolled in
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